DNA Methylation-dependent regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice.. Mus musculus

Global DNA hypomethylation in CD4+ cells in SLE patients was suggested to play a key role in the pathogenesis. To identify new methylation-sensitive genes, we integrated genome-wide DNA methylation and mRNA profiling in CD4+ cells of MRL/lpr (MRL) and C57BL6/J (B6) mice. We identified Ctse, in which 13 methyl-CpGs within 583 bp region of intron 1 were hypomethylated, and mRNA upregulated in MRL compared with B6 mice. One of methyl-CpGs, mCGCG was hypomethylated and mutated to CGGG in MRL mice. Kaiso is known to bind mCGCG and we hypothesized that it represses expression of Ctse. The binding of Kaiso to mCGCG site in B6 was reduced in MRL mice revealed by ChIP-PCR. EL4 cells treated with 5-azaC and/or TSA showed the suppression of the binding of Kaiso to mCGCG motif and the overexpression of Ctse was demonstrated by qPCR. Ctse gene silencing by siRNA in EL4 cells resulted in reduction of IL-10 secretion. Accordingly, IL10 and CTSE mRNAs up-regulated in CD4+ T cells both in MRL mice and the patients with SLE. The hypomethylation of mCGCG motif, reduced recruitment of Kaiso, and increased expression of Ctse and Il-10 in CD4+ cells may be involved in the pathogenesis of SLE. Overall design: To identify new candidate genes regulated by DNA methylation and involved in the pathogenesis of systemic lupus erythematosus (SLE), we performed Illumina Hiseq to analyze the genome-wide DNA methylation and expression of mRNA of CD4+ T cell isolated from spleens in MRL/lpr-Tnfrsf6lpr (MRL) mice and C57BL/6J (B6) mice. Both mice are female, 16 weeks old. For Illumina analysis, we used one mouse of MRL mice and one mouse of B6 mice.

已有研究表明，系统性红斑狼疮（SLE）患者CD4+细胞中的全基因组DNA低甲基化，在疾病发病机制中发挥关键作用。为鉴定新的甲基化敏感基因，本研究对MRL/lpr（简称MRL）小鼠与C57BL/6J（简称B6）小鼠的CD4+细胞进行全基因组DNA甲基化与mRNA表达谱整合分析。本研究鉴定到组织蛋白酶E（Ctse）基因：其内含子1的583bp区域内存在13个甲基化CpG位点，该区域在MRL小鼠中呈低甲基化状态，且其mRNA表达水平较B6小鼠显著上调。其中一个甲基化CpG位点mCGCG在MRL小鼠中既呈低甲基化状态，又突变为CGGG序列。已知Kaiso蛋白可结合mCGCG序列，因此我们推测Kaiso会抑制Ctse基因的表达。染色质免疫沉淀-聚合酶链反应（ChIP-PCR）结果显示，与B6小鼠相比，MRL小鼠中Kaiso与mCGCG位点的结合能力显著降低。用5-氮杂胞苷（5-azaC）和/或曲古抑菌素A（TSA）处理EL4细胞后，Kaiso与mCGCG基序的结合受到抑制，且实时定量聚合酶链反应（qPCR）证实Ctse基因表达显著上调。在EL4细胞中通过小干扰RNA（siRNA）沉默Ctse基因，可导致IL-10分泌量减少。相应地，IL10与CTSE的mRNA表达水平在MRL小鼠及SLE患者的CD4+ T细胞中均显著上调。CD4+细胞中mCGCG基序的低甲基化、Kaiso募集减少，以及Ctse与Il-10表达上调，可能参与了SLE的发病机制。实验设计：为鉴定受DNA甲基化调控、且参与系统性红斑狼疮（SLE）发病机制的新候选基因，本研究采用Illumina HiSeq测序平台，对MRL/lpr-Tnfrsf6lpr（简称MRL）小鼠与C57BL/6J（简称B6）小鼠脾脏分离得到的CD4+ T细胞进行全基因组DNA甲基化与mRNA表达谱分析。实验所用小鼠均为16周龄雌性小鼠，其中MRL小鼠与B6小鼠各1只用于Illumina测序分析。